Phospholipids are recognized as one of the most important classes of biological molecules. They are typically composed of a glycerol backbone which is acylated with fatty acids at the C.sub.1 and C.sub.2 positions and phosphorylated at the remaining terminus. The fatty acids may or may not be identical, and they may vary in degree of unsaturation. In one sub-class of phospholipids, the ether lipids, an ether linkage with an aliphatic chain is formed at the C.sub.1 position. Phospholipids are also typically esterified with head groups on the phosphate. Some of the commonly occurring head groups include ethanolamine (phosphatidylethanolamine), choline (phosphatidylcholine), glycerol (phosphatidylglycerol), serine (phosphatidylserine), and inositol (phosphatidylinositol).
Phospholipids have been shown to perform various indispensable cellular functions. They are the major structural components of cell membranes which separate the cytoplasm of the cell from the extracellular medium. Phosphatidylinositol has additionally been shown to be involved in both intracellular and intercellular signal transduction (Hawthorne, J. N., in Phospholipids: Biochemical, pharmaceutical and analytical considerations; I. Hanin and G. Pepeu, Ed.; Plenum Press: New York, 1990; pp 233-240). Dipalmitoyl phosphatidylcholine is an essential component of the pulmonary surfactant which reduces the surface tension in the alveolar membrane (B. Lachmann, ibid., pp 185-196). Platelet activating factor, an ether lipid, plays an important role in inflammation and asthma (Handley, D. A. and R. N. Saunders, Drug Dev. Res. 1986, 7, pp 361-375, and Snyder, F., Med. Res. Rev. 1985, 5, pp 107-140.). Furthermore, phospholipids have found widespread uses in food and cosmetic industries.
Various conjugates of phospholipids with other biologically active molecules have been described in the scientific literature. The phospholipid moiety generally imparts greater hydrophobicity to the conjugate and in some cases, promotes the incorporation of the conjugate onto or the transfer of the conjugate across the cell membrane. It is well known, for example, that muramyl tripeptide-phosphatidylethanolamine (MTP-PE) is active in vivo and is found in various organs 24 hours after injection whereas the parent peptide, muramyl dipeptide, shows activity in vitro and is excreted out of the body 60 minutes after injection (Fogler, W. E., R. Wade, D. E. Brundish, I. J. Fidler, J. Immunol. 1985, 135, pp 1372-1377, and Phillips, N. C., J. Rioux, M.-S. Tsao, Hepatology 1988, 8, pp 1046-1050). These results suggest that the absorption of the peptide is enhanced by conjugation to a lipophilic moiety. Other agents that have been coupled to phospholipids include acyclovir (Welch, C. J., A. Larsson, A. C. Ericson, B. Oberg, R. Datema, J. Chattopadhyaya, Acta Chem. Scand. 1985, B39, pp 47-54), ganglioside G.sub.M1 (Pacuszka, T., R. M. Bradley, P. H. Fishman, Biochemistry 30, pp 2563-2570), oligosaccharides (Childs, R. A., K. Drickamer, T. Kawasaki, S. Thiel, T. Mizuochi, T. Feizi, Blochem. J. 1989, 262, pp 131-138), serum transferrin (Azelius, P., E. J. F. Demant, G. H. Hansen, P. B. Jensen, Biochim. Biophys. Acta 1989, 979, pp 231-238), biotin, and fluorescent reagents.
Phospholipids are amphiphilic in nature and have a propensity to form micelles and bilayers in an aqueous medium. The bilayers also form closed vesicles called liposomes which have been used to encapsulate molecules of biological interest, including drugs, proteins, vitamins and dyes. Functionalized liposomes are being actively investigated as vehicles for targeted drug delivery. Galactosylated phospholipids, for example, have been incorporated in liposomes and used to deliver the liposomes specifically to asialoglycoprotein receptors of the hepatic system (Haensler, J. and F. Schuber, Glycoconjugate J. 1991, 8, pp 116-124). Immunoliposomes, constructed by covalent conjugation of antibodies to the phospholipid moieties on the liposomal surface, have also shown promise in targeting liposomes to specific cell tissues (Nassander, U. K., P. A. Steerenber, H. Poppe, G. Storm, L. G. Poels, W. H. De Jong, D. J. A. Crommelin, Canc. Res. 1992, 52, pp 646-653, and Pinnaduwage, P. and L. Huang, Biochemistry 1992, 31, pp 2850-2855).
Various methods have been described for derivatizing phospholipids to facilitate their conjugation with other molecules or moieties (for review, see Heath, T. D. and F. J. Martin, Chemistry and Physics of Lipids 1986, 40, pp 347-358). However, each of these methods suffers from various difficulties in practical application. For example, one method comprises glutaraldehyde activation of phosphatidylethanolamine and ultimate conjugation to amines by reductive amination. The problem of dimerization both between the phospholipids and between proteins has made this method less than ideal. An alternative method comprises amide formation between phosphatidylethanolamine and the carboxyl terminus of a peptide or protein. However, this method suffers from low yields and formation of byproducts.
In yet another approach, the phospholipid and the protein are first activated and then reacted to form the conjugate. For example, Hutchinson et. al. describe a method in which a phosphatidylethanolamine is activated with N-succinimidyl-S-acetyl-thioacetate (SATA) and treated with a hydroxylamine to yield a phospholipid-thiol derivative. The protein of interest is also activated with maleimide and then treated with the phospholipid derivative to form a stable conjugate via a thioether (Hutchinson et. al., FEBS Lett. 1986, 234, pp 493-6). In a variation of this protocol, the phosphatidylethanolamine is activated with a maleimido moiety and the lysine residue of a protein is activated with a protected thiol (Loughrey, H. C. et. al., J. Immun. Methods 1990, 132, pp 25-35). In practice, protocols employing these approaches are cumbersome to perform and the cost of the derivatizing agent is prohibitively expensive for scales above multigram quantities.
Phospholipid conjugates have also been formed by functionalizing phosphatidylethanolamine using a crosslinking reagent (e.g. dithiobis(succinimidyl propionate)) and reacting this intermediate with a lysine-containing protein so that the succinimidyl moiety is displaced by the amino group of the lysine residue (Afzelius, P., Biochem. Biophys. Acta 1989, 979, pp 231-8). However, crosslinking reagents are not economically feasible for producing phospholipid conjugates, particularly on a large scale. In yet another method, phosphatidylethanolamine may be coupled to glycosylated proteins via the protein carbohydrate chain. For example, glycans can be oxidized with sodium periodate to give reactive aldehydes which can then be coupled to phosphatidylethanolamine via reductive amination with sodium cyanoborohydride (Heath, T. et. al., Biochim. Biophys. Acta 1980, 599:42). This method is limited in its application only to glycoproteins and is often associated with low yields and byproduct formation.
None of the heretofore described methods offer a simple, generally applicable, efficient and economical (i.e. practical) means for generating phospholipid conjugates.